In all ensiling experiments, the amount of plant material available was severely limited due to working in glasshouse facilities. The quantities of wilted plant material used for individual vacuum bag silage packets (termed silos from herein) were 20g for measuring silage pH at intervals throughout the fermentations and 40-50g for detailed ‘silage quality’ and nutritional analysis. Both quantities are lower than has been previously reported in the literature for vacuum bag silos. The sizes chosen were a compromise between the amount of plant material available, modelling accuracy and the need to generate technical replicates for fermentation analyses over time. Due to the small size and number of silos, it was necessary to ensure careful pre- and post-harvest treatment of the material in order to ensure a minimal amount of variation in the material entering each silo. This was achieved through;
An adequate model silo design.
The use clonal PRG plants (in Experiment 5), randomized in space and shuffled around the glasshouse bench regularly during regrowth to minimize positional effects.
Harvesting homogenous leafy tissue, and largely excluding stem and dead material from silos. Pooling all the harvested regrowth from a given genotype/population*2 and then thoroughly
mixing the entire mass at key time points post-harvest.
The use of a commercial LAB inoculant to reduce the probability of unusual fermentations due to variation in natural or introduced microflora on leaf surfaces.
Using controlled rapid wilting procedures, to ensure a consistent DM between PRG materials within an experiment.
Even and accurate application of additives (commercial inoculant and later glucose).
*2 the terms genotype and population are both used because some experiments involved comparing the ensiling characteristics
of a PRG genotype with a PRG population (Appendix IV).
3.2.1 Model silo design
A double-bag model silo was used for Experiments 1-5. The design followed the method described in Hoedtke and Zeyner (2011) for vacuum packed plastic bag silages, with minor modifications. The desired amount of wilted and chopped PRG (+/- 0.5g), with or without additives, was placed in an ordinary plastic bag (Photograph 3.1, top left), and compressed by hand into one corner of the bag. The top of this bag was twisted and cut (Photograph 3.1, top right) and then the open end was taped down. The constrained herbage was further compressed and shaped by hand and then rubber bands were used to generate fairly uniform and spherical packets under compression (Photograph 3.1, bottom left). One rubber band was
used per 20g silo and two rubber bands per 50g silo. The bags were punctured with a disinfected needle to allow gas and effluent escape. The bags were placed into a 20 x 15 cm, 130 micron thick vacuum bag (Munro, Australia), from which air was evacuated with a vacuum packer (V.350, LAVA vacuum package, Bad Saulgau, Germany) (Photograph 3.1, bottom right). Using manual settings, the vacuum was allowed to reach the 50mbar maximum vacuum setting and was then left at this level of vacuum for 10 seconds (for 20g silos) or 20 seconds (for 50g silos), before triple heat sealing.
Advantages of silo design
Generating these double bagged spherical silos was considered to be advantageous because herbage was held in a single mass at a high density by the rubber bands for the duration of their fermentation, even when production of silo gases or effluent led to a loosening of the outer vacuum bag. Therefore compression was considered to represent packing density in a silage stack. Further, the shape minimized the surface area to volume ratio of the herbage within the silos.
3.2.2 Preliminary silo assessment
Prior to the major method development ensiling experiments (Experiments 1-4), the model silo was tested using paddock-grown perennial ryegrass. The model silo design was assessed primarily by pH readings of water extracts of ensiled material (as described in Chapter 3.6.5) and also by visual observation and volume measurements. Model silos of a given herbage weight were visually similar in size and in shape. Herbage was held at an approximate density of 0.5g.cm−3 (measured using the water displacement method) immediately after vacuum packing, a density comparable with stack silo densities recorded in the literature (0.32-0.64g cm−3) (Muck & Holmes, 2000; Muck et al., 2004). Accurate measurements of herbage density were not possible after this, due to gas production from fermentation. However, the inner bags could be seen to retain their shape and approximate volume throughout ensiling e.g. (Photograph 3.2).
Paddock-grown perennial ryegrass silage pH curve
The pH of water extracts of silage made from 50g and 20g paddock-grown PRG, wilted to approximately 30% DM, double-bagged, and vacuum-packed (with and without LAB inoculant) showed that well- preserved silage could be made using this method. Very low variation in pH occurred between duplicate silos, especially for inoculated material (Table 3.1). The data also indicated that the reduction in pH over time was similar for 20g and 50g paddock-grown PRG silage, providing that the same post-harvest treatments were applied and that care was taken in mixing plant material thoroughly at key post-harvest time points.
Table 3.1 pH of double-bagged, vacuum packed silos made from 20g and 50g wilted paddock-grown perennial ryegrass, with and without commercial inoculant. Values represent means ± SD (n = 2). Day 0 pH was estimated from the literature (Playne & McDonald, 1966).
Days of fermentation 20g 50g
+LAB -LAB + LAB -LAB
0 6.50 6.50 6.50 6.50
2 4.14 ± 0.01 6.31 ± 0.03 4.12 ± 0.07 6.20 ± 0.1
4 4.00 ± 0.07 4.85 ± 0.01 3.99 ± 0.01 4.95 ± 0.07
8 3.97 ± 0.07 4.55 3.96 ± 0.07 4.70 ± 0.06
30 3.95 ± 0.07 4.25 ± 0.03 3.93 ± 0.07 -
The following sections provide justification for the post-harvest treatments which were sequentially introduced during the method development phase (Experiments 1-4) in order to generate well-preserved silage from glasshouse-grown PRG. These post-harvest treatments, and the ensiling experiment in which they were introduced, were; LAB inoculant addition (Experiment 1), a controlled, rapid wilt (Experiment 1), and glucose addition (Experiment 4). Protocols for each post-harvest treatment are described.